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Cell, Tumor, and Stem Cell Biology |
1 Department of Biochemistry and Molecular Biology, 2 Medical Science and Engineering Research Center for Bioreaction to Reactive Oxygen Species, and 3 Biomedical Science Institute, School of Medicine, Kyung Hee University, Seoul, Korea
Requests for reprints: Wonchae Choe and Sung Soo Kim, Department of Biochemistry and Molecular Biology, School of Medicine, Kyung Hee University, 1, Hoegi-dong, Dongdaemoon-gu, Seoul 130-701, Korea. Phone: 822-961-0940, 822-961-0524; Fax: 822-959-8168; E-mail: wchoe{at}khu.ac.kr and sgskim{at}khu.ac.kr.
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Abstract |
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transcription factor. Interestingly, overexpression of CypA prevented hypoxia- and cisplatin-induced apoptosis, and this was associated with the suppression of reactive oxygen species generation and depolarization of mitochondrial membrane potential, whereas small interfering RNA–based CypA knockdown aggravated these factors. These results suggest that CypA is important in tumorigenesis, especially in tumor apoptosis. [Cancer Res 2007;67(8):3654–62] |
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Although little is known about the function of CypA in cancer cells, it has been recently reported that CypA is overexpressed in many cancer cells, including human pancreatic cancer cells (8, 9), oral squamous cancer cells (10, 11) and non–small cell lung cancer (12, 13). Recent reports have shown that CypA interacts with the retinoblastoma susceptibility gene product (Rb) in Jurkat T cells (14). Although the role of CypA in apoptosis is not clear at this point, several reports show that CypA is released from cardiac myocytes in response to hypoxia/reoxygenation and may protect cardiac myocytes from oxidative stress–induced apoptosis (15), and that CypA also protects other cells from oxidative stress–induced apoptosis (16, 17). These reports suggest that CypA might be important for tumorigenesis in solid tumors.
To elucidate the role of overexpressed CypA in cancer cells, we examined CypA induction under hypoxic conditions and investigated the effect of CypA on hypoxia- or cisplatin-induced cell death. In this study, we show, for the first time, that CypA is transcriptionally induced by hypoxia-inducible factor-1 (HIF-1) transcription factor. Furthermore, we show that overexpressed CypA in cancer cells protects cells against cellular stresses, including hypoxia and cisplatin treatment, at least in part as a result of its antioxidant function.
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Materials and Methods |
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Reverse transcription-PCR. For the analysis of CypA mRNA, total RNA was prepared from cells using the TRIzol reagent (Invitrogen, San Diego, CA). Total RNA (2 µg) was amplified by a two-step protocol using the avian myeloblastosis virus reverse-transcriptase (Promega, Madison, WI) and Taq polymerase. Primers for amplifying CypA transcripts were as follows: forward, 5'-GCAAGCTTACCATGGTCAACCCCACC-3'; reverse, 5'-GCGGATCCGAGTTGTCCACAGTCGGA-3'. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amplified as an internal control for reverse transcription-PCR (RT-PCR). Amplified products were separated on 1% agarose gel.
Construction of plasmids for CypA promoter analysis. The CypA promoter sequence was analyzed using MatInspector of Genomatrix.4 Four hypoxia-responsive element (HRE) candidates were identified in the CypA promoter. For the construction of luciferase reporter plasmids, 1,012 bp of the CypA promoter sequence were serially deleted by 200 bp through PCR. The deleted fragments were cloned into pGL3 basic vector (Promega) with KpnI and HindIII. For mutational analysis, each putative HRE site on the CypA promoter was mutated by PCR-based site-directed mutagenesis. Primers used for constructing luciferase reporters containing various regions of the CypA promoter or mutated HRE sites are as follows:
Luciferase assay. NIH3T3, DU145, and HeLa cells were transfected with 0.5-µg amounts of the pGL3 basic-derived plasmids, together with internal control plasmid pSV-ß-gal (Promega). The cells were exposed to hypoxia for 12 h and harvested in 150 µL of lysis buffer [1% Triton X-100, 25 mmol/L Gly-Gly, 15 mmol/L MgSO4, and 2 mmol/L EGTA (pH, 8.0)]. Luciferase and ß-gal activities (data not shown) were measured using 20 µL of each cell lysate using a microplate reader (Bio-Rad, Richmond, CA), and luciferase activity was normalized based on ß-gal activity, as reported previously (18).
Preparation of nuclear extracts and electrophoretic mobility shift assay. Nuclear extracts were prepared according to previous reports (19–21). Electrophoretic mobility shift assay was done with oligonucleotides (CypA-WT, 5'-CCTTTTATACCACGTTCGGC CCCG-3'; CypA-M, 5'-GCCCCTTTTATACTATATTCGGCCCCGCCC-3') derived from the CypA promoter sequences. Sense and antisense strands of oligonucleotides were annealed into double-stranded oligonucleotides and were labeled with [
-32P] ATP (Amersham Biosciences, Buckinghamshire, United Kingdom). Nuclear extracts were obtained from DU145 and NIH3T3 cells after hypoxic incubation for 12 h. The nuclear extracts (10 µg) were incubated in the presence of 5x binding buffer [25% glycerol, 50 mmol/L Tris-HCl (pH, 7.5), 250 mmol/L NaCl, 5 mmol/L DTT, 1 mg/mL poly(deoxyinosinic-deoxycytidylic acid), 5 mmol/L EDTA]. For the interaction of the nuclear extracts and radiolabeled oligonucleotides, the reaction mixture was incubated for 30 min at room temperature. For a competition study, a 100-fold molar excess of unlabeled oligonucloetides was added to the reaction mixture before the addition of the radiolabeled probe. The samples were run on a 5% nondenaturing polyacrylamide gel. The gels were then dried and exposed to X-ray film with an intensifying screen at –80°C.
MTT assay. Cell viability was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) conversion assay in a 12-well plate. The culture medium was replaced with 1 mL medium containing 0.5 mg/mL MTT (Sigma-Aldrich, St. Louis, MO) and incubated for 60 min at 37°C. The blue-colored tetrazolium crystals resulting from mitochondrial enzymatic activity on the MTT substrate were solubilized with 150 µL DMSO. The absorbance was read at 595 nm in a microplate reader (Bio-Rad). Cell survival was expressed as the percentage of absorbance relative to that of untreated cells.
Cell cycle analysis. Cells were grown to 60% confluence and treated with 2 mmol/L thymidine for up to 24 h to arrest the cells in the S phase. The cells were washed in PBS and released in fresh medium for specified periods of time. For cell cycle analysis, cells were harvested, rinsed with PBS, and fixed in 70% ethanol overnight at 4°C. The fixed cells were centrifuged and resuspended in 1 mL of PBS containing 0.05 mg/mL propidium iodide and 0.2 mg/mL RNase-A and then incubated at 4°C for 1 h. DNA content was determined by FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ) to analyze the cell cycle.
Analysis of ROS and mitochondrial membrane potential (
m) measurement. Reactive oxygen species (ROS) were measured using 2'-7'-dichlorodihydrofluorescein diacetate (DCF-DA, Molecular Probe) dye. Human prostate carcinoma DU145 cells were grown to 80% confluence in complete media and treated with and without cisplatin (30 µmol/L). Then, the cells were loaded with 10 µmol/L DCF-DA at 37°C for 30 min and resuspended in 1 mL of PBS. Fluorescence was measured by a flow cytometer (FACSCalibur, Becton Dickinson). The mean DCF fluorescence intensity was measured with excitation at 488 nm and emission at 525 nm. Untreated cells were used as a reference for ROS levels. The mitochondrial membrane potential was measured using 3, 3-dihexyloxacarbocyanine iodide dye (DiOC6; Molecular Probes, Eugene, OR). After treatment with cisplatin (30 µmol/L), cells were loaded with 40 nmol/L DiOC6 for 40 min at 37°C and resuspended in 1 mL of PBS before the flow-cytometric analysis. DiOC6 was excited at 488 nm, and fluorescence was analyzed at 525 nm.
Subcellular fractionation. Cells were suspended in lysis buffer [250 mmol/L sucrose, 0.1 mmol/L EDTA, 2 mmol/L HEPES (pH, 7.4)], homogenized, and centrifuged at 900 x g for 10 min. Then, the supernatants were recentrifuged at 10,000 x g for 20 min. The resulting supernatants (cytosol) were subjected to 15% SDS-PAGE and analyzed by immunoblotting with anti–cytochrome c oxidase subunit IV (COX4) antibody (Clontech, Palo Alto, CA) to confirm that the cytosolic fraction was successfully separated from the mitochondrial fraction (data not shown).
Hoechst staining. Cells were incubated for 30 min with Hoechst 33342 (Molecular Probes) loading dye and fixed for 20 min in 4% formaldehyde. After washing with ice-cold PBS thrice, stained cells were monitored using the confocal laser microscope (META 510, Zeiss, Thornwood, NY). Apoptotic cells were identified by nuclear condensation and fragmentation.
Stable transfection. pcDNA3-CypA/WT (3 µg) or pcDNA3 (3 µg) alone was transfected into DU145 cells using GenePORTER (Genlantis, San Diego, CA). Cells used for stable transfection were cultured in the selective medium with 600 mg/mL G418 for a month. Then, drug-resistant individual clones were isolated and transferred to a six-well plate for further amplification in the presence of selective medium.
Small interfering RNA. Small interfering RNAs (siRNA) specific to either CypA (CypA-siRNA) or control sequence (control-siRNA) were prepared by Eurogene tech (Intron Biotechnology, Kyungki, Korea). siRNA (0.5 µg) was transfected into cells using METAFECTENE transfection reagent (Biontex, Munich, Germany). siRNA target sequences were as follows: CypA-siRNA (sense, 5'-UGACUUCACACGCCAUAAUdTdT-3'; antisense, 5'-AUUAUGGCGUGUGAAGUCAdTdT-3') and control-siRNA (universal negative control). The efficiency of siRNA-based interference of CypA was monitored by immunoblot.
Recombinant adenovirus. CypA/WT cDNA was cloned into pCA14. For homologous recombination, pCA14-CypA was transformed into Escherichia coli BJ 5183 along with vmRL-H5dl 324Bst. Recombinant adenovirus was propagated in HEK 293 cells. The viral DNA constructs were confirmed by HindIII restriction enzyme digestion (data not shown). Adenovirus stocks were purified through a cesium gradient for 4 h (444,000 x g, 10°C) and titrated using a standard plaque assay. Cells were infected with recombinant adenovirus at a multiplicity of infection (MOI) of 300 for 24 h to express exogeneous CypA. Uninfected or infected cells with adeno-GFP (Ad) were used as a negative control for CypA expression.
Immunoblotting. Cell extracts were separated by SDS-PAGE and transferred onto a nitrocellulose membrane. After blocking, the membrane was incubated with the indicated primary antibody, followed by incubation with a secondary antibody. Samples were detected with enhanced chemiluminescence reagents (Santa Cruz Biotechnology, Santa Cruz, CA). Unless specified, actin protein was immunoblotted to standardize the amount of sample proteins for the immunoblot analysis. Antibodies against CypA, PARP, cytochrome c, or actin were obtained from Santa Cruz Biotechnology and Upstate Biotechnology (Lake Placid, NY).
Statistical analysis. Results were expressed as means ± SE from at least three independent experiments. Statistical analysis was done by using Student's t test. Unless otherwise indicated, P < 0.05 was deemed significant.
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Results |
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transcription factor. Up-regulation of CypA by hypoxia led us to explore whether the CypA promoter has a HRE, the HIF-1 binding site. First, bioinformatic analysis of the promoter of the human CypA gene was done to search for a consensus HRE sequence 5'-CGTG-3'. We identified four putative HRE sequences located at –47 bp (HRE1), –77 bp (HRE2), –309 bp (HRE3), and –589 bp (HRE4) upstream to the CypA start codon (Fig. 2A
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is known to be a major transcription factor in hypoxia (22). Thus, we overexpressed HIF-1 by transfection of pcDNA3-HIF-1 and compared the luciferase activity of the transfectants in normoxia with that of controls to determine whether HIF-1 induces the transcription of CypA. As in the hypoxia experiments, all luciferase constructs containing a functional HRE2 showed about a 2-fold increase in luciferase activity when pcDNA3-HIF-1 was cotransfected (Fig. 2B). Interestingly, pGL3-CypA-200 displayed a low luciferase activity under normoxic conditions, compared with other CypA luciferase constructs (Fig. 2B). In addition, pGL3-CypA-200 showed a weaker basal luciferase activity, compared with pGL3-CypA-1012 in the HIF-1 transfection experiment, indicating that other cis-acting elements required for the general expression of CypA are located upstream of HRE2. We finally located a putative GC box at –317 bp from the starting codon of CypA (data not shown).
Next, because CypA has been reported to be expressed more in tumor than in normal cells (8–13), the luciferase activity of pGL3-CypA-1012 was measured in cancer cells, such as DU145 and HeLa cells. As shown in Fig. 2C, the luciferase activity was increased by 3.5- and 3-fold in these cancer cells by hypoxia and HIF-1 transfection, respectively. Taken together, these data suggest that HIF-1 regulates CypA expression in response to hypoxia.
Binding of HIF-1 to the HRE sequence of CypA promoter. To determine if HIF-1 binds to the HRE2 sequence of the CypA promoter, EMSA experiments were done using nuclear extracts from DU145 cells incubated for 12 h under hypoxic conditions. Oligonucleotides containing wild-type (WT) or mutated version of HRE2 (see Materials and Methods) were used in the experiments. First, we tested whether HIF-1 binds to the HRE2 sequences of the CypA promoter under normoxic conditions. 32P-labeled oligonuleotides containing WT HRE2 were complexed with nuclear protein extracts from DU145 cells cultured under normoxic conditions, whereas the 32P-labeled oligonuleotides containing mutated HRE2 were not (Fig. 2D, lanes 1 and 2). The complex was more easily observed when cultured under conditions of hypoxia, whereas oligonuleotides containing mutated HRE2 did not form complexes (Fig. 2D, lanes 4 and 5). The specificity of this complex was verified by competitive inhibition with an excess of the same unlabeled oligonucleotide (Fig. 2D, lanes 3 and 6). We verified the results by reproducing consistent EMSA data with nuclear extracts from NIH3T3 (data not shown). Therefore, we concluded that up-regulation of CypA is indeed mediated by HIF-1 in response to hypoxia.
CypA desensitizes cells to hypoxia- and cisplatin-induced cell death by suppression of ROS increase and loss of mitochondrial membrane potential. HIF-1 has been known to play an important role in growth and survival of solid tumor cells (23, 24). We have shown that CypA is induced under hypoxic conditions via the HIF-1 transcription factor. Therefore, we hypothesized that CypA might play an important role in growth and survival of solid tumor cells, especially in response to cellular stress including hypoxia, cisplatin, and oxidative stress such as H2O2 treatment.
First, to assess the role of CypA in hypoxia-induced cell death, we did a MTT assay after DU145 cells were stably transfected with mock vector, pcDNA3-CypA/WT or transiently transfected with CypA-siRNA and were exposed to hypoxia for up to 4 days. Before the hypoxic experiments, the expression level of exogenous CypA was evaluated in the transfected cells by immunoblotting (Fig. 3A ). Compared with endogenous CypA, exogenous CypA showed an almost 2-fold increase in expression. In addition, the expression of CypA was almost 95% suppressed by specific siRNA interference (Fig. 3A). As presented in Fig. 3B, CypA transfectants exposed to hypoxia for 4 days showed significantly higher cell survival rates than mock transfectants; CypA knockdown showed the highest cell death rate among the transfectants, indicating that CypA protects cells from hypoxia-induced cell death.
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Cytoprotective roles of CypA against apoptosis. The effects of hypoxia, cisplatin, and H2O2 treatment on the processing of apoptosis-related protein PARP and cytochrome c release from mitochondria to cytoplasm in CypA transfectants and CypA knockdown were investigated using immunoblotting. Incubation of cells under hypoxic conditions or treatment with cisplatin or H2O2 showed cleavage of the caspase-3 substrate, PARP, as indicated by the appearance of the 85-kDa cleaved PARP product (Fig. 4A and B ). Accordingly, the extent of cytochrome c release was also increased with cellular stress. As expected, overexpression of CypA reduced the processing of PARP and cytochrome c release (Fig. 4A). By contrast, CypA knockdown increased the processing of PARP and cytosolic release of cytochrome c significantly, compared with control-siRNA (Fig. 4B). In addition, suppression of CypA in cisplatin-induced apoptosis was verified by Hoechst staining (Fig. 4C). These data show that CypA overexpression desensitizes responses of apoptosis-related proteins to hypoxia, cisplatin, and H2O2, suggesting that overexpressed CypA in cancer cells protects cells by attenuating stress-induced apoptosis.
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CypA does not affect cellular proliferation. Recently, it has been proposed that CypA might enhance tumor growth by affecting cellular proliferation (12). To examine this hypothesis in our cell lines, we monitored the effects of CypA on cell cycle progression and on the doubling time of cells. Mock, CypA transfectant, and CypA knockdown were grown, synchronized by 2 mmol/L thymidine, and released in fresh medium for the specified periods of time and then subjected to fluorescence-activated cell sorting (FACS) analysis to determine DNA content. As presented in Fig. 6A , normal cell cycle progression was observed in each cell line. We could not see any difference in DNA content among mock, CypA transfectants, and CypA knockdown over time up to 16 h. In addition, there was no difference in the doubling time (Fig. 6B). Their doubling time was about 15 h. These data suggest that the cellular function of CypA may be related to apoptosis, rather than cellular proliferation, in our tested cell lines.
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Discussion |
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under hypoxic conditions, a microenvironment in solid tumors. We also investigated the role of overexpressed CypA in cancer cells. In this paper, we showed that transcription of the CypA was induced by hypoxic conditions through a mechanism that involves binding of HIF-1 to the HRE2 site located at –77 within the CypA promoter. We reproduced these results using a variety of cell lines, including DU145 and HeLa cells. Therefore, induction of CypA in response to hypoxia is not limited to a specific cell line. Interestingly, luciferase activity of pGL3-CypA-200 was lower than that of pGL3-CypA-1012 (Fig. 2). This result suggests that the promoter region from –1,012 to –201 might have other cis-acting elements required for general transcriptional induction. In fact, we found a putative GC box (–317 bp) in the region (data not shown). Because a functional GC box is a positive regulatory element in the promoter, the putative GC box is possibly responsible for the high constitutive expression of CypA gene.
Cancer cells are more resistant to stress than normal nontransformed cells. They use multiple defense systems against a variety of stresses, including hypoxia, radiation, and anticancer drugs. Under hypoxic conditions, cancer cells can survive by inducing HIF-1 expression (10, 22). Therefore, HIF-1 has been suggested as a potential regulator of hypoxia-induced apoptotic cell death (23). Induction of apoptosis by hypoxia in HIF-1 transfected cells has been shown to be more difficult than in cells that do not express HIF-1. This led us to speculate that the resistance of HIF-1 transfected cells to hypoxia-induced cell death may be, at least in part, due to the ability of HIF-1 to up-regulate CypA expression in cells; this was because our data showed that hypoxia induced expression of CypA through direct activation of HIF-1, and that the induced CypA seemed to resist cellular stresses, such as hypoxia insult, cisplatin treatment, and oxidative stress. In addition, our results also showed that CypA knockdown aggravated cell death induced by hypoxia, cisplatin treatment, and oxidative stress in the tested cell lines. Because a cytoprotective role of CypA was also observed even in p53-defective cells, CypA may be a good target for new chemotherapeutic drug development to alleviate cisplatin resistance frequently observed in p53-defective cancer patients.
Interestingly, overexpression of CypA did not offer any resistance to other anticancer drugs such as paclitaxel and 5-fluorouracil (data not shown). Considering that apoptosis induced by these drugs is not directly related to the increased ROS, although ROS are generated after treatment with these drugs (28, 29), we thought that the suppression of the increased ROS generation may be involved, at least in part, in the cytoprotection of overexpressed CypA against cellular stresses, such as hypoxia and cisplatin treatment; this hypothesis turned out to be true in this case (Fig. 3C and D). Accordingly, evidence that CypA functions as an antioxidant has been recently accumulated (16, 30–32). Our previous study also showed that overexpressed CypA removed ROS generated by cyclosporin A (17). Moreover, CypA has been reported to be secreted in response to ROS in vascular smooth muscle cells (33). Therefore, it seems that the antioxidant role of CypA is at least partly critical for cytoprotection of overexposed CypA against hypoxia and cisplatin treatment. We propose here that the antioxidant function of CypA might require PPIase activity because the CypA R55A mutation, which is defective in PPIase activity, also aggravated ROS generation and depolarization of the mitochondrial membrane (data not shown). Consistent with our proposal, cyclosporin A, a well-known inhibitor of PPIase activity in CypA, has been reported to have chemotherapeutic effects in a variety of cancer cells (34, 35). The molecular mechanism of CypA as an antioxidant should be pursued in future studies.
Recently, Howard et al. reported on the importance of CypA to tumor growth in vivo, specifically on proliferation and apoptosis (12). We also investigated the possibility that CypA may influence proliferation by monitoring cell cycle progression and doubling time of CypA transfectants and CypA knockdown samples. We found no difference in cellular proliferation among mock, CypA transfectants, and CypA knockdown samples. Our data are consistent with a recent report showing that loss of CypA in mouse embryonic stem cells had no detectable effect on cell growth or viability (36). The discrepancy between our findings and those of Howard et al. (12) concerning the involvement of CypA in proliferation is not understood at this time. Therefore, we cannot rule out the possibility that CypA might affect cellular proliferation in specific cell lines.
In conclusion, our data suggest that CypA may play a critical role in tumorigenesis by reducing apoptosis both under hypoxic conditions and with cisplatin treatment. The cytoprotective role of CypA seems to require PPIase activity. Therefore, the PPIase activity of CypA may be a good chemotherapeutic target for treatments that could facilitate to alleviate the chemoresistance in tumor cells including p53-defective tumors. We continue to study CypA to further understand its cytoprotective mechanisms, which will hopefully lead to new chemotherapeutic strategies against solid tumors.
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Acknowledgments |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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Footnotes |
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Received 5/15/06. Revised 11/18/06. Accepted 1/ 3/07.
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overexpression indicates a good prognosis in early stage squamous cell carcinomas of the oral floor. BMC Cancer 2005;5:84.[CrossRef][Medline] in hypoxia-mediated apoptosis, cell proliferation and tumour angiogenesis. Nature 1998;394:485–90.[CrossRef][Medline] inhibits tumor growth in hepatobiliary and pancreatic cancers. Cancer Gene Ther 2006;13:131–40.[CrossRef][Medline]This article has been cited by other articles:
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  F. T. Bane, J. H. Bannon, S. R. Pennington, G. Campaini, D. C. Williams, D. M. Zisterer, and M. M. Mc Gee The Microtubule-Targeting Agents, PBOX-6 [Pyrrolobenzoxazepine 7-[(dimethylcarbamoyl)oxy]-6-(2-naphthyl)pyrrolo-[2,1-d] (1,5)-benzoxazepine] and Paclitaxel, Induce Nucleocytoplasmic Redistribution of the Peptidyl-Prolyl Isomerases, Cyclophilin A and Pin1, in Malignant Hematopoietic Cells J. Pharmacol. Exp. Ther., April 1, 2009; 329(1): 38 - 47. [Abstract] [Full Text] [PDF]
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 J. Kim, T. G. Choi, Y. Ding, Y. Kim, K. S. Ha, K. H. Lee, I. Kang, J. Ha, R. J. Kaufman, J. Lee, et al. Overexpressed cyclophilin B suppresses apoptosis associated with ROS and Ca2+ homeostasis after ER stress J. Cell Sci., November 1, 2008; 121(21): 3636 - 3648. [Abstract] [Full Text] [PDF]
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 Z. Li, X. Zhao, S. Bai, Z. Wang, L. Chen, Y. Wei, and C. Huang Proteomics Identification of Cyclophilin A as a Potential Prognostic Factor and Therapeutic Target in Endometrial Carcinoma Mol. Cell. Proteomics, October 1, 2008; 7(10): 1810 - 1823. [Abstract] [Full Text] [PDF]
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) or without (
) 30 µmol/L cisplatin for 24 h. Then, ROS was measured by flow cytometry after loading with DCF-DA. D, measurement of mitochondrial membrane potential. Each transfectant was treated with (+) or without (–) 30 µmol/L cisplatin for 48 h. Then, the cells were incubated with DiOC6 and subjected to flow-cytometric analysis. Arrows, mitochondrial membrane potential depolarized cells.
